Journal Of Pharmaceutical And Biomedical Analysis[JOURNAL]
Sun
200 papers
AI-assisted integrative framework combining microarray data analysis and cerebrospinal fluid pharmacology for revealing molecular mechanism in Alzheimer's disease: A case study of Psoraleae Fructus.
Li X, Qi J, Pan F … +9 more , Zhang Y, Wang L, Yi Z, Liu H, Zheng J, Pan Y, Liu F, Jiang L, Chen S
J Pharm Biomed Anal · 2026 Oct · PMID 42166920 · Publisher ↗
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a gradual decline in cognitive function, with a complex pathogenesis involving multiple targets and signaling pathways, and current th...
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a gradual decline in cognitive function, with a complex pathogenesis involving multiple targets and signaling pathways, and current therapeutic strategies remain insufficient to achieve satisfactory outcomes. Psoraleae Fructus (PF) has been reported to exhibit potential therapeutic effects against AD. However, its multi-target mechanisms of action have not yet been systematically elucidated. In this study, an AI-assisted integrative computational and experimental framework was established to comprehensively investigate the molecular basis of PF intervention in AD. Targeted constituents of PF were first identified using UPLC-Q Exactive Orbitrap high-resolution mass spectrometry, followed by the integration of Artificial intelligence modeling, microarray data, and network pharmacology to screen hub targets. Cerebrospinal fluid-based pharmacological assays, together with molecular docking, molecular dynamics simulations, microarray validation, and western blot, were subsequently employed to validate. The results demonstrated that PF markedly modulated the expression of GSK3β and PPARγ, thereby regulating core AD-related pathological markers, including p-tau, Aβ, β-secretase, and inflammatory mediators. Collectively, this study delineated a multi-target regulatory network underlying the anti-AD effects of PF and provided a robust theoretical foundation for its further translational investigation.
Comparison of feature-based molecular networking strategies for untargeted metabolite identification in endophytic fungal samples from Lespedeza bicolor Turcz.
Liu Y, Zheng Q, Liu Q … +3 more , Tao Y, Huang H, Ding B
J Pharm Biomed Anal · 2026 Oct · PMID 42160990 · Publisher ↗
Feature-Based Molecular Networking (FBMN) is a robust strategy for the structural elucidation of natural products; however, inconsistent workflows across software platforms and ionization modes hinder its standardized ap...
Feature-Based Molecular Networking (FBMN) is a robust strategy for the structural elucidation of natural products; however, inconsistent workflows across software platforms and ionization modes hinder its standardized application. This study systematically evaluated six FBMN construction approaches using LC-MS/MS data acquired from secondary metabolites produced by Lespedeza bicolor-associated endophytic fungi. We compared the MZmine and GNPS (Global Natural Products Social Molecular Networking, https://gnps.ucsd.edu) platforms across positive and negative ionization modes, as well as across different data integration strategies. The results indicated that platform selection and ionization polarity significantly influenced network topology and annotation efficiency. The MZmine-based data-level merged network exhibited superior performance in node density and overall metabolite coverage. Conversely, the GNPS-based network-level merging strategy was more effective in grouping unknown metabolites and facilitating structural interpretation. Positive ionization consistently yielded a higher number of annotations and better annotation accuracy than negative mode, while dual-polarity integration significantly enhanced network connectivity. Specifically, GNPS network-level merging strengthened spectral-similarity linkages, whereas MZmine data-level merging maximized the representation of chemical diversity. These findings demonstrate that a combinatorial approach, integrating dual ionization modes and complementary FBMN platforms, is essential for optimizing metabolite identification in complex natural product matrices.
Systematic characterization of active components and therapeutic mechanisms of Chaihu Guizhi Ganjiang Decoction in metabolic dysfunction-associated steatohepatitis.
Wu H, Wang T, Cai S … +4 more , Qiao Y, Liu J, Li Y, Xiao H
J Pharm Biomed Anal · 2026 Oct · PMID 42160989 · Publisher ↗
Chaihu Guizhi Ganjiang Decoction (CGGD) is a clinically proven prescription effective against metabolic dysfunction-associated steatohepatitis (MASH). However, its chemical constituents with the lipid-lowering and anti-i...
Chaihu Guizhi Ganjiang Decoction (CGGD) is a clinically proven prescription effective against metabolic dysfunction-associated steatohepatitis (MASH). However, its chemical constituents with the lipid-lowering and anti-inflammatory bioactivities and underlying mechanisms remain unclear. This study aimed to characterize the chemical profile of CGGD while elucidating their lipolytic and anti-inflammatory activities and mechanisms. Using UHPLC-Q-TOF-MS/MS, 209 compounds were identified in CGGD, categorized into 52 saponins, 103 flavonoids, 17 gingerols, 21 organic acids, and 16 others, with 13 putative unvalidated new compounds. Nineteen prototype components were detected in hepatic tissues, with saikosaponin A, baicalin, wogonoside, skullcapflavone II, and glycyrrhizin exhibiting strong binding affinities toward PPARα and TLR4 proteins. These components significantly reduced triglyceride (TG) accumulation in FFA-induced HepG2 steatosis cells, confirmed by oil red O staining and TG quantification. Mechanistically, saikosaponin A and baicalin upregulated PPARα and its downstream genes (CPT1A, FABP1, ACOX1) to promote fatty acid transport and oxidation. Meanwhile, saikosaponin A, baicalin, and wogonoside significantly suppressed mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in LPS-stimulated RAW264.7 macrophages, and saikosaponin A and wogonoside further inhibited the expression of TLR4, MyD88, and phosphorylated NFκB, thereby attenuating inflammation. In conclusion, this study established a comprehensive methodology for profiling the chemical constituents of CGGD and identified saikosaponin A, baicalin, wogonoside, skullcapflavone II, and glycyrrhizin as its key anti-MASH components. Their lipid-lowering and anti-inflammatory activities were mediated through the PPARα-regulated fatty acid metabolism pathway and the TLR4/MyD88/NFκB signaling pathway, respectively, offering a scientific basis for the clinical application of CGGD in MASH.
Quantification of PEG2000-DMG and TS-202, an ionic lipid, in rabbit plasma by LC-MS/MS: Application to complement C5 siRNA lipid nanoparticles.
Mano Y, Hotta K
J Pharm Biomed Anal · 2026 Oct · PMID 42160988 · Publisher ↗
A simple and robust assay was developed for the quantification of PEG2000-DMG and TS-202, key components of the lipid nanoparticle (LNP) formulation of E8001-LNP, which encapsulates small-interfering RNA targeting comple...
A simple and robust assay was developed for the quantification of PEG2000-DMG and TS-202, key components of the lipid nanoparticle (LNP) formulation of E8001-LNP, which encapsulates small-interfering RNA targeting complement C5, in rabbit plasma. The assay utilizes high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To prevent degradation of PEG2000-DMG, plasma samples were treated with a serine protease inhibitor prior to protein precipitation extraction. The analytes were separated on a reversed-phase column under gradient elution conditions. Detection was performed using selected reaction monitoring in positive ion mode, with mass transitions of m/z 780.8 → 698.9 for PEG2000-DMG and m/z 650.5 → 126.3 for TS-202. The quantification ranges were 0.05-5 µg/mL for PEG2000-DMG and 0.5-50 µg/mL for TS-202, with validated dilution integrity up to 100-fold. Accuracy and precision of quality control samples at five concentration levels met acceptance criteria, remaining within ±15%. Stability studies confirmed the bench-top, freeze-thaw, and long-term frozen stability of both analytes in rabbit plasma. Application to a toxicokinetic study in rabbits revealed that PEG2000-DMG was eliminated much faster than TS-202. The incurred sample reanalysis further confirmed the assay reproducibility.
Development and validation of a GC-MS method for the simultaneous determination of bisphenols, phthalate monoesters, and triclosan in human meconium for comprehensive prenatal exposure assessment.
Tok KC, Gumustas M, Masoud KM … +3 more , Arsan S, Bolkan AG, Suzen HS
J Pharm Biomed Anal · 2026 Oct · PMID 42155372 · Publisher ↗
Determining in utero exposure to endocrine-disrupting chemicals (EDCs) is crucial for public health, given their widespread presence and high production volumes. Analyzing EDCs, such as bisphenols, phthalate monoesters,...
Determining in utero exposure to endocrine-disrupting chemicals (EDCs) is crucial for public health, given their widespread presence and high production volumes. Analyzing EDCs, such as bisphenols, phthalate monoesters, and triclosan in meconium is essential for evaluating prenatal exposure and gaining critical insights into potential fetal health hazards throughout gestation. The present work describes a GC-MS method for the determination of bisphenols, phthalate monoesters, and triclosan in meconium samples. Sample preparation involved hydrolysis of glucuronidated analytes followed by liquid-liquid back extraction with dichloromethane. A BSTFA+ 1% TMCS was used for derivatization of compounds prior to the GC-MS analysis. The method was linear over the concentration range 0.25-10 ng/g for all analytes. Intra- and inter-day precision (%RSD) values ranging from 0.39% to 5.79% and 1.07-6.97%, respectively. Accuracy (%) was within 85-115%. Application to real human meconium samples revealed that bisphenols were frequently detected, particularly BPS and BPA, with BPS detected in 96.0% of the samples (0.88-24.98 ng/g) and BPA in 88.0% (0.32-7.72 ng/g), while BPF and BPAF were found less frequently, in 36.0% and 24.0% of the samples, respectively. Among the phthalate monoesters, MMP, MEP, and MEHP were ubiquitous, while MBP was detected in nearly all samples (96.0%). Triclosan was also quantified, though at a lower frequency (12.0%). These findings confirm the prenatal exposure to these EDCs and demonstrate that the developed method provides the sensitivity and robustness required to quantify these contaminants at environmentally relevant concentrations.
Monitoring disulfide scrambling of monoclonal antibody biologics using liquid chromatography-mass spectrometry coupled to online electrochemical reduction.
Huang L, Zhao Y, Mao Y … +1 more , Li N
J Pharm Biomed Anal · 2026 Oct · PMID 42155371 · Publisher ↗
Electrochemical flow-cell (EC) devices have been used to perform online reduction of disulfide bonds found in both peptides and full-length proteins. This study demonstrates the use of a liquid chromatography-EC-mass spe...
Electrochemical flow-cell (EC) devices have been used to perform online reduction of disulfide bonds found in both peptides and full-length proteins. This study demonstrates the use of a liquid chromatography-EC-mass spectrometry (LC-EC-MS) platform to monitor disulfide scrambling in the therapeutic monoclonal antibody (mAb), casirivimab. As a first step, disulfide scrambling was artificially induced during enzymatic digestion at pH 8.5. EC-assisted online partial reduction was performed via a post-column reduction scheme. Retention time (RT) alignment was used to validate the composition of the parental disulfide linkage. Fingerprints of all potential disulfide-linked pairs were generated based on the results of partial reduction with no need for additional information on any of the specific parental species. The performance of partial reduction was evaluated by varying critical parameters, including EC-cell voltage and the sample loading amount. The improved performance allows us to capture low-level disulfide scrambling such as linkages involving the hinge region. Additionally, the LC-EC-MS platform is used to monitor site-specific disulfide scrambling progress under high pH stress condition. Samples with varying levels of disulfide scrambling were prepared by alkylating free cysteines at different time points during digestion to perform a time-course study. As a result, we successfully monitored scrambling level of 13 sites through MS1-level relative quantification as well as their free thiol species. Low level of disulfide scrambling across the casirivimab was captured at 0.5 hr time point. Overall, we demonstrated that the online LC-EC-MS platform provided us with significant insights into patterns of disulfide scrambling.
Illuminating biomarkers: Metal-based strategies and fluorescence nanosensors for clinical monitoring.
Magdy G, Ali MAM, Elattar RH … +5 more , Radwan AS, Abdel-Hakim A, Chaudhary AA, El-Maghrabey M, Kishikawa N
J Pharm Biomed Anal · 2026 Oct · PMID 42155370 · Publisher ↗
Plasmonic and fluorescence nanosensors have become vital tools for recent diagnostic applications due to their great sensitivity, rapid response, and capacity to determine very small amounts of biological targets. Plasmo...
Plasmonic and fluorescence nanosensors have become vital tools for recent diagnostic applications due to their great sensitivity, rapid response, and capacity to determine very small amounts of biological targets. Plasmonic sensors rely on localized surface plasmon resonance (LSPR), which occurs when metallic nanostructures, such as gold or silver nanoparticles, generate strong electromagnetic fields on their surfaces. This phenomenon enables sensors to detect changes produced by biomarker or pathogen binding with great accuracy and precision. Recent advances in nanoparticle design and surface modification have improved their selectivity and appropriateness for point-of-care diagnostic devices. On the other hand, fluorescence nanosensors measure changes in fluorescence wavelength or intensity when a target analyte interacts with a fluorescent probe. Advanced fluorescent nanomaterials, including quantum dots, carbon dots, MOF-based fluorophores, and organic dyes, have significantly increased signal stability, biocompatibility, and detection limits. These developments have allowed for the sensitive determination of disease-related analytes such as nucleic acids, proteins, and metabolites. Furthermore, approaches like aggregation-induced emission and fluorescence resonance energy transfer (FRET) have improved the capabilities of fluorescence-based sensing platforms. Both plasmonic and fluorescence nanosensors provide reliable, fast, and simple diagnostic options for early illness diagnosis and clinical monitoring. This review focuses on the operating principles of both approaches, current advances in material design, and their expanding importance in diagnostic applications. This review provides a thorough resource that can successfully support and progress further research and developments in the field, particularly with regard to the applications of comparable nanosensors.
Corrigendum to "Characterizing the metabolites of the tyrosine-kinase inhibitor pexidartinib in mouse feces, urine, plasma, and liver" [J. Pharm. Biomed. Anal. 265 (2025) 117034].
Qin X, Chen S, Hakenjos JM … +6 more , Wang J, Guo L, Dogra A, Hu Z, MacKenzie KR, Li F
J Pharm Biomed Anal · 2026 Oct · PMID 42140031 · Publisher ↗
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Illuminating the Dark Host Cell Proteome: A host cell protein coverage method for LC-MS impurity assays.
Chrone VG, Kofoed M, Draborg AH … +4 more , Schwämmle V, Kofoed T, Højrup P, Mørtz E
J Pharm Biomed Anal · 2026 Sep · PMID 42128475 · Publisher ↗
Host cell protein (HCP) analysis by liquid chromatography-mass spectrometry (LC-MS) enables identification and quantification of individual impurities in biopharmaceutical products, supporting risk-based impurity assessm...
Host cell protein (HCP) analysis by liquid chromatography-mass spectrometry (LC-MS) enables identification and quantification of individual impurities in biopharmaceutical products, supporting risk-based impurity assessment. However, unlike enzyme-linked immunosorbent assay (ELISA) methods, systematic evaluation of method-specific HCP coverage for LC-MS assays remain poorly defined. In particular, limited attention has been given to proteins that are inherently undetectable under specific analytical conditions, potentially creating blind spots in impurity characterization. Here, we present DARK-COV, a transparent and generalizable in silico pipeline for assessing theoretical HCP coverage of LC-MS-based impurity assays. The pipeline combines physicochemical filtering of in silico-digested proteomes-based on peptide hydrophobicity, charge state, and mass-to-charge ratio-with assay-specific LC-MS parameters to predict which peptides and proteins can be detected. Proteins predicted to yield fewer than two detectable peptides were classified as Dark Host Cell Proteins. The pipeline was benchmarked against a large-scale experimental dataset derived from more than 15,000 LC-MS analyses curated in HCPedia™, a comprehensive database of empirically observed HCP peptides generated using the same LC-MS workflow. As a proof-of-concept demonstration, DARK-COV was applied to four industrially relevant expression systems-Chinese Hamster Ovary (CHO) cells, Escherichia coli, Human Embryonic Kidney (HEK) cells, and Spodoptera frugiperda (Sf9) cells-revealing that only 1.0-5.2% of proteins across these proteomes were predicted to be theoretically undetectable under idealized in silico conditions. These predictions present an upper bound on detectability and do not account for matrix effects, ion suppression, or concentration-dependent detectability in real samples. Comparison with experimental observations across > 15,000 LC-MS datasets demonstrated strong concordance with DARK-COV predictions, supporting the classification of proteins that are likely to fall outside the detectable range of the LC-MS assay. Functional annotation and comparison against known high-risk HCPs indicated that the majority of Dark Host Cell Proteins are low-molecular-weight proteins with limited relevance to biopharmaceutical impurity risk. Overall, this pipeline provides a reproducible, method-specific approach for quantifying theoretical coverage and comparing predictions with experimental observations in LC-MS assays. By identifying analytical blind spots, the strategy supports risk-based evaluation of undetected HCPs and offers a rational foundation for regulatory justification of the LC-MS-based HCP impurity analyses described in USP Chapter 1132.1.
Identification, toxicity evaluation, and HPLC-based quality control method development of process-related impurities and degradation products of belumosudil mesylate.
Wang Y, Wu Z, Han Z … +3 more , Li Z, Zhang T, Li L
J Pharm Biomed Anal · 2026 Oct · PMID 42127555 · Publisher ↗
As an oral small-molecule drug that selectively inhibits ROCK2, Belm exhibits unique advantages in the treatment of hormone-dependent/refractory cGVHD. However, impurity control and stability during the production of its...
As an oral small-molecule drug that selectively inhibits ROCK2, Belm exhibits unique advantages in the treatment of hormone-dependent/refractory cGVHD. However, impurity control and stability during the production of its API remain key challenges. This study conducted a systematic investigation into Belm's synthetic process, impurity analysis, toxicity evaluation, and quality control. First, the synthetic route was optimized to avoid the environmental hazards and low-yield issues of traditional routes. Nine relate impurities (including 5 new impurities: Imp-B, Imp-D, Imp-E, Imp-F and Imp-I) were successfully synthesized. An HPLC-based method for the separation and detection of Belm and its impurities was established, and method validation, Confirmed its applicability for API quality control. Impurity structures were elucidated by MS and NMR, and forced degradation studies (under acidic, basic, oxidative, thermal and photolysis conditions) were performed to reveal Belm's degradation behavior. Its mesylate form shows good stability under acidic and thermal conditions but is prone to forming new impurities (Imp-D1, Imp-D3 and Imp-D4) under basic, oxidative, and photolysis conditions. Toxicity prediction of impurities using ProTox-II and ADMETlab 3.0 indicated that Imp-D possesses significant immunotoxicity. Further network pharmacology analysis and molecular docking clarified the potential immunotoxicity mechanism of Imp-D. This study provides critical data support for the optimization of Belm's production process, formulation of impurity control strategies, and guarantee of stability. It also lays a foundation for its drug registration applications and full-life-cycle quality management.
Sensitive and selective quantification of elcatonin in equine plasma and urine using LC-FAIMS-MS/MS.
Ohnuma K, Fukazawa M, Uchida T … +5 more , Kato T, Sugai-Bannai M, Shibuya M, Yamada M, Hirano-Kodaira M
J Pharm Biomed Anal · 2026 Oct · PMID 42127554 · Publisher ↗
The use of drugs for enhancing bone strength is prohibited under international regulations governing horse racing and equestrian sports. Elcatonin, a synthetic peptide with analgesic and bone-strengthening properties, is...
The use of drugs for enhancing bone strength is prohibited under international regulations governing horse racing and equestrian sports. Elcatonin, a synthetic peptide with analgesic and bone-strengthening properties, is routinely monitored for compliance. However, its cyclic structure and high molecular weight hinder reliable detection. In this study, we developed and validated a sensitive liquid chromatography-high-field asymmetric waveform ion mobility spectrometry-tandem mass spectrometry method for quantifying elcatonin in equine plasma and urine. Sample preparation was optimised via weak cation exchange solid-phase extraction. A pilot pharmacokinetic and pharmacodynamic study was conducted to confirm the practical applicability of our method by validating linearity, reproducibility, sensitivity, selectivity, recovery, and analyte stability, including enzymatic/proteolytic stability. The method achieved a detection limit of 10 pg/mL for urine and plasma (R > 0.9995 in both cases), meeting the validation criteria for quantitative analysis. Following administration, elcatonin was detected in plasma (for up to 6 h) but not in urine. Pharmacodynamic analysis revealed that elcatonin administration caused a transient small decrease in the plasma calcium levels without any other observable clinical effects, such as hypocalcaemia. The established method overcomes the challenges of detecting and quantifying cyclic peptides and has the potential to improve the detectability of elcatonin and related molecules relevant to doping control and animal healthcare.
A validated ligand binding assay for the determination of etalanetug, a novel anti-tau therapeutic antibody, in rats and monkeys for nonclinical pharmacokinetic studies.
Aoyama M, Noritake Y, Kita K … +1 more , Mano Y
J Pharm Biomed Anal · 2026 Oct · PMID 42127553 · Publisher ↗
Etalanetug (E2814) is a novel human monoclonal antibody targeting tau that is currently being developed as a biotherapeutic for Alzheimer's disease. To inform pharmacological and toxicological evaluations in nonclinical...
Etalanetug (E2814) is a novel human monoclonal antibody targeting tau that is currently being developed as a biotherapeutic for Alzheimer's disease. To inform pharmacological and toxicological evaluations in nonclinical settings and to guide dose selection for the first-in-human study, it is critical to characterize the pharmacokinetics (PK) of etalanetug. We have developed and validated a ligand-binding assay (LBA) for quantifying etalanetug in rat and monkey serum for use in nonclinical PK studies. Tau protein and anti-human immunoglobulin G antibody served as the capture and detection reagents, respectively. Using 10 µL of serum at a minimum dilution of 2000, etalanetug was quantifiable from 0.1 µg/mL in monkeys and 0.3 µg/mL in rats. Assay accuracy and precision, together with multiple serum stability assessments, demonstrated the robustness and reliability of the method. The assay was applied to PK studies in rats and monkeys. Following an intravenous dose of 30 mg/kg, etalanetug exhibited a biphasic decline in serum concentration with half-lives of 150-193 h in rats and monkeys. Incurred sample reanalysis further confirmed assay reproducibility. These results support the suitability of the developed LBA for nonclinical PK studies of etalanetug in both rats and monkeys.
Investigation of excipient-related artifacts observed in the leachables study of tiragolumab by LC-HRMS and GC-MS.
Du J, Pearson R, Zhou Y … +1 more , Skidmore K
J Pharm Biomed Anal · 2026 Sep · PMID 42119499 · Publisher ↗
Leachables studies of protein formulations are faced with many analytical challenges arising from relatively high levels of complex formulation components. In the stressed sample of the monoclonal antibody drug tiragolum...
Leachables studies of protein formulations are faced with many analytical challenges arising from relatively high levels of complex formulation components. In the stressed sample of the monoclonal antibody drug tiragolumab with the main excipients methionine, histidine, sucrose, and polysorbate, two unknown compounds were detected in the screening for non-volatile leachables by reversed phase (RP) liquid chromatography high-resolution mass spectrometry (LC-HRMS). Unknown 1 was proven to be a gas-phase mass spectral adduct between methionine and the sucrose-derived hexose. Separation of methionine and hexose by hydrophilic interaction chromatography (HILIC) resulted in the disappearance of the unknown peak. It was also observed that non-covalent amino acid-sugar adducts in the gas phase were prevalent in both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) modes. Unknown 2 was identified as a Maillard reaction product 1-(3H-imidazo[4,5-c]pyridin-4-yl)ethan-1-one by LC-HRMS and gas chromatography mass spectrometry (GC-MS). The formation mechanism was confirmed by the reaction between histidine and the sucrose-derived methylglyoxal (MGO) under hydrothermal conditions. This study exemplifies false positive leachable results and delves into the chemical reactions and interactions that generated such artifacts.
Comprehensive impurity profiling and stability assessment of indacaterol maleate integrating LC-MS and in silico genotoxicity.
Aydın Ç, Yazar Y, Yılmaz H … +2 more , Bellur Atici E, Özkan SA
J Pharm Biomed Anal · 2026 Sep · PMID 42119498 · Publisher ↗
This study describes the development and validation of stability-indicating analytical methods for indacaterol maleate, an ultra-long-acting β-adrenoceptor agonist used in asthma and chronic obstructive pulmonary disease...
This study describes the development and validation of stability-indicating analytical methods for indacaterol maleate, an ultra-long-acting β-adrenoceptor agonist used in asthma and chronic obstructive pulmonary disease (COPD). RP-HPLC-UV methods were established for quantifying indacaterol and its related substances, while LC-QDa-MS and high-resolution LC-QTOF-MS were employed for impurity identification and structural elucidation. All methods were developed and validated in accordance with ICH guidelines to support comprehensive assessment of process-related impurities and degradation behavior. Eight potential impurities were identified, synthesized, and fully characterized. The validated RP-HPLC-UV assay and related substances methods were applied to forced degradation studies under thermal, photolytic, oxidative, neutral, acidic, and alkaline conditions, as well as to accelerated and long-term stability samples. Trace-level impurities were structurally elucidated using LC-QDa-MS and LC-QTOF-MS. In silico mutagenicity assessment of thirteen impurities using Nexus software classified IND-B ((R)-8-(benzyloxy)-5-(2-bromo-1-hydroxyethyl)-quinolin-2(1H)-one) as a Class 3 potential genotoxic impurity due to its alkyl bromide moiety. Although the TTC-based limit for IND-B (based on the ICH M7 threshold of 1.5 µg/day for a maximum daily dose of 300 µg) corresponds to ≤ 0.5%, all specified impurities were conservatively controlled at ≤ 0.15% and unspecified impurities at ≤ 0.10%, in line with ICH M7 and Q3A guidelines. The RP-HPLC-UV assay and related substances methods demonstrated specificity, precision, accuracy, linearity, and robustness, confirming their suitability as stability-indicating methods. Overall, this integrated approach provides a robust framework for impurity profiling, stability assessment, and genotoxic risk evaluation of indacaterol maleate, ensuring regulatory compliance and product quality throughout shelf life.
Reconsidering astatine-211 analytics: Retention effects in reversed-phase HPLC.
Humpert S, Mues L, Pedersen NB … +3 more , Neumaier F, Herth MM, Neumaier B
J Pharm Biomed Anal · 2026 Sep · PMID 42105597 · Publisher ↗
Astatine-211 is a short-lived alpha-emitting radionuclide with high potential for cancer treatment by targeted alpha therapy (TAT). Reversed-phase high-performance liquid chromatography (RP-HPLC) is central to the develo...
Astatine-211 is a short-lived alpha-emitting radionuclide with high potential for cancer treatment by targeted alpha therapy (TAT). Reversed-phase high-performance liquid chromatography (RP-HPLC) is central to the development and quality control of At-labeled radiopharmaceuticals. However, accurate analysis remains challenging due to the ultra-trace levels of astatine and its complex, unpredictable chemical behaviour. In this study, we systematically evaluated the recovery of representative inorganic astatine formulations under different redox conditions using various chromatographic conditions, which is a prerequisite for reliable quantification. Examination of four different columns with distinct stationary phase chemistry demonstrated that significant discrepancies can arise when radiochemical conversion is assessed solely based on eluted activity, as is common standard for RP-HPLC analysis. Among the chromatographic conditions examined, the highest and most consistent astatine recoveries (88-98%) were achieved using a basic mobile phase containing 0.4% triethylamine in combination with a base-tolerant stationary phase, likely due to a shift in astatine speciation under alkaline conditions, reduced secondary interactions with the stationary phase, and/or potentially beneficial ion pairing effects. In contrast, widely used standard solvent systems based on acetonitrile/water with or without 0.1% trifluoroacetic acid resulted in unsatisfactory and highly variable recoveries (7-71% or 30-79%, respectively) across all columns investigated. These findings highlight the necessity of optimized chromatographic conditions and suggest that inadequate recovery of free astatine could lead to substantial misestimation of the radiochemical conversion and purity of radiopharmaceutical formulations if not explicitly accounted for by auxiliary quantification strategies.
Differences in hepatic metabolism of Liandan Xiaoyan Formula between control and ulcerative colitis mice associated with FXR/PXR-CYP450 changes.
Zheng B, Chen Y, Fan B … +7 more , Dai P, Zhou R, Li S, Liang L, Lin C, Liu F, Zhu C
J Pharm Biomed Anal · 2026 Sep · PMID 42105596 · Publisher ↗
Understanding the metabolic process of traditional Chinese prescription (TCP) during disease states and its underlying mechanisms is crucial for evaluating therapeutic efficacy and safety. Cytochrome P450 (CYP450)-mediat...
Understanding the metabolic process of traditional Chinese prescription (TCP) during disease states and its underlying mechanisms is crucial for evaluating therapeutic efficacy and safety. Cytochrome P450 (CYP450)-mediated hepatic metabolism plays a key role in this process. Liandan Xiaoyan Formula (LDXYF), a Chinese herbal prescription used to treat enteritis, exhibits different pharmacokinetic characteristics and metabolic profiles in ulcerative colitis (UC). To elucidate its underlying metabolic mechanisms, UPLC-Q-Exactive Orbitrap MS/MS technology was employed to compare the metabolite profiles of its six main effective components in hepatic microsomes from control versus UC mice. Molecular docking was employed to predict the dominant CYP450 isoforms involved. Subsequently, a CYP450 enzyme selective inhibition assay was used to identify the metabolic enzyme phenotypes for each component. Finally, RT-qPCR and Western blot analyses were conducted to confirm the expression changes of key isoforms and their regulatory targets. Metabolite profiling revealed that the biotransformation of LDXYF components was markedly suppressed in UC mice, with hydrogenation, carboxylation, hydroxylation, and demethylation being the most attenuated. The CYP450 phenotypes were identified as: CYP2D22/2C29 for andrographolide; CYP2C29 for dehydroandrographolide; CYP2D22/2C29 for 14-deoxyandrographolide; CYP2C29 for 1-methoxycarbonyl-β-carboline; CYP2D22/2C29 for 4,5-dimethoxycanthin-6-one; and CYP2C29 for 5-hydroxy-4-methoxycanthin-6-one. Furthermore, hepatic CYP2D22 and CYP2C29 were confirmed to be down-regulated at both mRNA and protein levels in UC, which correlated with reduced expression of the bile-acid receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR) and the transcription factor hepatocyte nuclear factor 4 alpha (HNF4α). In conclusion, UC is associated with suppressed LDXYF metabolism and reduced hepatic CYP450 expression, accompanied by altered FXR/PXR-related signaling, thereby providing mechanistic insight into drug-disease interactions.
Degradation study of thiamine hydrochloride and analysis of expired ampoules using a novel, validated HPLC-UV method.
Klapper S, Holzgrabe U
J Pharm Biomed Anal · 2026 Sep · PMID 42105595 · Publisher ↗
Thiamine is a vital vitamin and one-sided diet carries a high risk of deficiency. Supplementation is simple with appropriate pharmaceutical products. Unfortunately, the availability of medicines is still not guaranteed e...
Thiamine is a vital vitamin and one-sided diet carries a high risk of deficiency. Supplementation is simple with appropriate pharmaceutical products. Unfortunately, the availability of medicines is still not guaranteed everywhere in the world, while at the same time, perfectly good medicines are being disposed of after their expiry date. For this reason, the shelf-time has often been critically questioned in recent years. Analysing finished pharmaceutical products, stored for decades under uncontrolled conditions, offers a rare insight in real-world stability of pharmaceuticals. Hence, this study examined 80-year-old thiamine hydrochloride ampoules. For comparison, test solutions were stored at 60 °C for 40 days in order to induce degradation reactions. A novel HPLC-UV method was developed and validated in accordance with ICH Guideline Q2(R2). Gradient elution, using a hydrophilic end-capped C18 column (150 × 4.6 mm, particle size 5 µm) with eluent A water/formic acid (100/0.1 v/v) and eluent B acetonitrile/formic acid (100/0.1 v/v) from 0% B to 90% B under UV detection (254 nm), enables the separation of thiamine, 6 degradation products, the preservatives methyl- and propylparaben and their degradation product p-hydroxybenzoic acid. The method enables the structural elucidation of degradation products using high-resolution tandem mass spectrometry experiments. A significant degradation of thiamine hydrochloride, with a residual content of 30.1, 37.2, and 44.8%, was observed. In addition to the expected degradation products, resulting from hydrolysis, further degradation products were formed, that were not expected in the given acidic range. Stress samples showed the same degradation profile as the expired ampoules.
Multi-omics reveals the role of PPARα signaling in the treatment of hyperlipidemia with Qinlian Hongqu Decoction.
Gan Q, Zhang Z, Yue Y … +7 more , Song W, Zhao M, He Y, Luo Y, Yang Y, Dang S, Zhang Y
J Pharm Biomed Anal · 2026 Sep · PMID 42096839 · Publisher ↗
Qinlian Hongqu Decoction (QLHQD), a traditional Chinese herbal formula, exerts therapeutic effects on high-fat diet (HFD)-induced lipid metabolism disorders, but its underlying mechanisms remain unclear. This study aimed...
Qinlian Hongqu Decoction (QLHQD), a traditional Chinese herbal formula, exerts therapeutic effects on high-fat diet (HFD)-induced lipid metabolism disorders, but its underlying mechanisms remain unclear. This study aimed to investigate QLHQD's efficacy in treating hyperlipidemia and clarify its potential mechanisms. The chemical components of QLHQD were identified by ultra-performance liquid chromatography (UPLC). A hyperlipidemic rat model was established via HFD feeding, and the effects of QLHQD on serum lipids, hepatic steatosis, inflammation, oxidative stress, and gut microbiota were evaluated using automatic biochemical analysis, enzyme-linked immunosorbent assay (ELISA), hematoxylin-eosin (HE) staining, oil red O staining, reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry (IHC), and 16S rDNA amplicon sequencing. Transcriptomics, network pharmacology, metabolomics, molecular docking, and molecular dynamics simulations were integrated to explore mechanisms, with key proteins validated by Western blot (WB) and IHC. Seven QLHQD components were identified; animal experiments showed QLHQD reduced serum lipids, hepatic steatosis, inflammation, and oxidative stress, and regulated gut microbiota in hyperlipidemic rats. Integrated transcriptomics and network pharmacology revealed significant enrichment of the peroxisome proliferator-activated receptor α (PPARα) signaling pathway, and molecular docking confirmed strong affinity between all seven QLHQD components and PPARα protein. WB, IHC, and metabolomics verified that QLHQD likely ameliorates HFD-induced dyslipidemia and hepatic steatosis by regulating bile acid metabolism via the PPARα pathway. In conclusion, QLHQD effectively alleviates HFD-induced dyslipidemia and hepatic steatosis in hyperlipidemic rats, with the underlying mechanism possibly associated with regulating the PPARα-bile acid metabolism pathway.
Integrated metabolomics and transcriptomics analysis to reveal the mechanism of Xuefu Zhuyu Decoction in the treatment of nitroglycerin-induced chronic migraine.
Zhu TT, Wang ZJ, Ren KK … +5 more , Liu WD, Xiao LJ, Feng YL, Yang SL, Ouyang H
J Pharm Biomed Anal · 2026 Sep · PMID 42090750 · Publisher ↗
Migraine is a chronic neurological disorder. As a classic formula for promoting blood circulation and removing blood stasis, Xuefu Zhuyu Decoction (XFZYD) has shown definite clinical efficacy in the treatment of migraine...
Migraine is a chronic neurological disorder. As a classic formula for promoting blood circulation and removing blood stasis, Xuefu Zhuyu Decoction (XFZYD) has shown definite clinical efficacy in the treatment of migraine with blood stasis syndrome; however, its biological mechanisms have not yet been fully elucidated and warrant further investigation. Therefore, in this study, we employed untargeted metabolomics, combined with transcriptomic sequencing, to identify endogenous differential metabolites in plasma and differentially expressed genes in brain tissue that were significantly regulated by XFZYD in migraine rats. We further explored the key targets and potential therapeutic mechanisms involved. Through integrated multi-omics analysis, the MAPK/ERK signaling pathway was ultimately identified as the key pathway regulated by XFZYD. Molecular biology experiments further confirmed that XFZYD modulated the expression of genes involved in inflammation, vascular function, and stress response within this pathway, acting on key genes such as COX-2, P-ERK1/2, Nr4a1, and Egr2, thereby intervening in two core pathological processes: vascular dysfunction and neurogenic inflammation. In summary, from both the transcriptomic and terminal metabolic levels, this study systematically elucidated the multidimensional mechanisms by which XFZYD treats migraine by reversing the "blood stasis" state and exerting its effect of "promoting blood circulation and removing blood stasis," thereby providing new research directions and a theoretical basis for its clinical application.
Hydrophilic interaction chromatography and tandem mass spectrometry for the characterization of methylated oligoribonucleotides.
Adouairi K, Farre C, Chaix C … +2 more , Pfeffer S, Faure K
J Pharm Biomed Anal · 2026 Sep · PMID 42090749 · Publisher ↗
Chemical modifications on oligonucleotides, notably N-6-methyladenosine (mA) are known for their impact on diverse classes of RNA including micro (mi)RNAs. However, the characterization of these modifications on intact s...
Chemical modifications on oligonucleotides, notably N-6-methyladenosine (mA) are known for their impact on diverse classes of RNA including micro (mi)RNAs. However, the characterization of these modifications on intact sequences remains challenging. An ion-pair free hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry (HILIC-HRMS/MS) workflow was developed for the intact analysis of miRNA analogues within synthesis batches, with length up to 22 nucleotides, including sequences differing by an increase in methylation levels and positional isomers containing the same number of methyl groups. The method combines amide-based HILIC separation to top-down collision-induced dissociation (CID) MS/MS and delivers three levels of characterization in a 20-min single run. It allows (i) the separation and identification of impurities (shortmers) from full length product (FLP), (ii) the chromatographic resolution of global methylation forms (0, 1 and 2 methyl groups) up to 22-mer sequence length and (iii) positional isomers discrimination based on the presence and/or absence of specific fragments. The application of HILIC-MS/MS workflow on complex samples, such as positional isomers with identical mA counts and non-equimolar mixtures of various methylation levels, demonstrated robust identification of each isomer, including in mixed samples and despite differences in isomer concentration.
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